gene therapy. Touraj Aligholipour Farzani, Seval Bilge Dagalp and Aykut Ozkul. Department of Veterinary Virology, Veterinary Faculty, Ankara University, Turkey.
Bovine herpesvirus type 4-BAC as an attractive viral vector for vaccination and gene therapy Touraj Aligholipour Farzani, Seval Bilge Dagalp and Aykut Ozkul Department of Veterinary Virology, Veterinary Faculty, Ankara University, Turkey
ABSTRACT
BHV-4 is a member of the family of Herpesviridae, subfamily Gammaherpesvirinae in the Rhadinovirus genus, and it is found worldwide among cattle populations (1, 2). According to the previous researches, Bovine herpesvirus type 4(BHV-4) is sparkling memberof the attractive new emerging viral vectors in the vaccination and gene therapy fields. Several biological characteristics of bovine herpesvirus 4 (BHV-4) make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types coming from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells (3).
In addition, compared to other herpesviruses BHV-4 has less complex genome with some determined gene areas to insert the desired gene. Being episomal in the target cells including macrophages,B and T cells makes this virus as a new candidate in the field of viral vectors (4). To develop a BHV-4-BAC vector in this research, we have selected the gene area among gp3 and gp4 which has been previously shown to besuitable for introduce of foreign DNA and after insertion of the BAC cassette the resulting virus is stable and able to replicate in vitro and in vivo(5). For this, BAC cassette from pBeloBAC11vector that has been ligated to CMV-EGFP-Neo cassette (from pEGFP-C1) flanked by loxp has been inserted between gp3 (Bo2) and gp4 (Bo3) of Movar33/63 strain of BHV-4. The extrachromosomal homologous recombination has been performed in the MDBK cell line afterelectroporation of circular plasmid containing homologous arms and BAC cassette after inoculation of the virus. After three rounds of G418 selection in BEK cells (700µg/ml) and one plaque purification, the recombinant virus DNA
has been extracted by Hirt method and used for transformationofDH10beta cells by electroporation(6). After 20 serial passages in the bacteria, the stability of BHV-4-BAC has been proved. This BHV-4-BAC vector system will be utilized in the future researches including viral vaccination and gene therapy.
Image
Figure1a.Construction of shuttle vector containing the homologous arms(Bo2 and Bo3) and loxp-BAC-CMVEGFP-Neo-loxp cassette
Figure1b.Insertion of loxp-BAC-CMV-EGFP-Neo-loxp cassette between gp3(Bo2) and gp4(Bo3) of BHV4 by extrachromosomal recombination in MDBK cells
References
1) Roizman, B., Desrosiers, R. C., Fleckenstein, B., Lopez, C., Minson, A.C. and Struddert, M.J. (1992).The familyherpesviridae: an update. Archives of Virology.123, 425-449.
2) Zimmermann, W.,BrollandH., Ehlers.(2001).Genomesequence of bovine herpesvirus 4 a bovine Rhadinovirus and identification of an origin of DNA replication. J. Virol. v.75, p.1186-1194.
3) Donofrio, G.,Cavirani, S., Vanderplasschen, A., Gillet, L. andFlammini, C,F.(2006). Recombinant Bovine Herpesvirus 4 (BoHV-4) ExpressingGlycoprotein D of BoHV-1 Is
Immunogenic and ElicitsSerum-NeutralizingAntibodiesagainst BoHV-1 in a rabbit Model. Clinical and Vaccine Immunology.
4) O. J. Lopez, J. A. Galeotaand and F. A. Osorio.(1996). BovineHerpesvirus type-4 (BHV-4) persistentlyinfectscells of themarginalzone of spleen in cattle. Microbial Pathogenesis.21: 47–58.
5) G. Donofrio, C. Sartori, V. Franceschi, A. Capocefalo, S. Cavirani, S. Taddei, and C.F. Flammini.(2008). Doubleimmunizationstrategywith a BoHV-4-vectorialized secretedchimericpeptide BVDV-E2/BoHV-1-gD. Vaccine26.6031–6042.
6) Hirt B. (1967). Selective extraction of polyoma DNA frominfected mouse cell cultures. J Mol Biol. 26:365–369.
